Enhanced gene expression in algae

ABSTRACT

The invention provides eukaryotic unicellular algae engineered to express a nucleosome alteration protein fused to a protein with affinity to the DNA binding site acting in coordination. An example is a LexA-p300 fusion protein, where the p300 is derived from  Chlamydomonas . The LexA binding domain guides the p300 to the binding site and the p300 loosens the nucleosome structure by acetylating histones within proximity of the transgene, thus remodeling the local chromatin structure to allow for high-level expression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/504,741, entitled “ENHANCED GENE EXPRESSION IN ALGAE” filed Apr. 27,2012, which issued as U.S. Pat. No. 8,476,019, on Jul. 2, 2013, andwhich is a national phase of PCT/US2010/055012, entitled “ENHANCED GENEEXPRESSION IN ALGAE” filed Nov. 1, 2010, which claims the prioritybenefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No.61/256,921, entitled “ENHANCED GENE EXPRESSION IN ALGAE” filed Oct. 30,2009, which are both incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of molecular biology and inparticular to the expression of transgenes in algae.

2. Description of the Background

Transgenes are foreign DNA sequences introduced into genomes, in thecase of eukaryotic cells within the chromosomes. These genes are usuallytranscribed as any other gene of the host. Transcription is generallycontrolled by the chromatin structure that packs the chromosome's DNAinto tight bundles in eukaryotic organisms called nucleosomes. As thechromatin structure around a specific gene relaxes, the DNA of theparticular gene becomes accessible to the transcription machinery of thecell. Staining indicates that actively transcribed genes in eukaryotesare more loosely incorporated in nucleosomes and more prevalent ineuchromatin. In some instances, transgenes are incorporated into thehost's chromosome but fail to be expressed due to unfavorable chromatinstructures. This phenomenon is called “gene silencing.” The ability tocontrol how tightly a nucleosome is packed can help enhance theexpression of transgenes in host cells. In mammalian cells, it has beenproposed that coupling transgene expression with increased availabilityof a histone “tail” modifying gene, p300 (also known as a histone acetyltransferase, or “HAT”; in the family of CREB binding proteins, or“CBP”), can increase the expression level, presumably because the acetyltransferase activity causes a looser histone-DNA association and allowstranscription factors access to the genes. T. H. J. Kwaks et al., J.Biotechnology, 115:35-46 (2005).

Microalgae encompass a broad range of organisms, mostly unicellularaquatic organisms. The unicellular eukaryotic microalgae (includinggreen algae, diatoms, and brown algae) are photosynthetic and have anucleus, mitochondria and chloroplasts. The chromatin structure in algaeis distinct from other eukaryotes. The chromatin in algae stainsheavily, indicating a more compact nucleosome structure and tightassociation of the DNA to the histones. These differences in chromatinstructure of microalgae, particularly in green algae, suggest distinctmechanism of histone chromatin regulation of gene expression.

These differences in eukaryotic microalgae chromatin structure may bethe factor behind the observation that stable nuclear transgeneexpression in microalgae is difficult and transient due to chromatinmediated gene silencing. H. Cerutti, A. M. J., N. W. Gillham, J. E.Boynton, Epigenetic silencing of a foreign gene in nuclear transformantsof Chlamydomonas, The Plant Cell 9:925-945 (1997). When geneticconstructs comprising a mammalian derived anti-apoptotic gene and afluorescent reporter gene were previously introduced by the presentinventors in algae, the expression levels were low and no expression ofthe fluorescence gene was detected, thus confirming that transgenes aredifficult to express in algae.

Algae are considered an important source of healthy nutrients for humanconsumption and are important as biomass and biofuels. Geneticengineering and stable (over multiple generations) expression oftransgenes would open new horizons and greatly enhance the value anddesirability to beneficially culture algae. However, as noted above,stable and sufficiently high level of gene expression has been difficultto achieve. A method to improve transgene expression in algae and makethat expression stable would be very useful. Such an approach would needto account for the uniquely robust histone mediated gene silencing ofmicroalgae including green algae.

SUMMARY OF THE INVENTION

In accordance to one embodiment, the invention provides a system forenhanced gene expression in algae, the system comprising:

-   -   an algae compatible transcriptional promoter functionally        upstream of a coding sequence for a gene expression enhancer        (GEE) fusion protein, wherein the fusion protein comprises an        algae derived p300 functionally fused to the DNA binding        protein, wherein at least the portion of the coding sequence of        the DNA binding protein domain is codon optimized for improved        expression in an algae;    -   at least one transgene functionally downstream of an algae        compatible transcriptional promoter; and    -   at least one DNA region that is a binding site for the DNA        binding protein, in vicinity of at least one of said        transcriptional promoters;    -   wherein said system resides in an algae.

In a preferred embodiment, the DNA binding protein is LexA DNA Bindingdomain. In another preferred embodiment, the p300 part of the GEE fusionprotein is from Chlamydomonas reinhardtii. In a more preferredembodiment, only a HAT domain of the p300 protein is part of the GEEfusion protein. The p300 or only the HAT domain of p300 may be derivedfrom homologs of other microalgae including green algae in addition toChlamydomonas reinhardtii.

In accordance to another embodiment, the transgene is codon modified forimproved expression in algae. In a preferred embodiment, the transgeneor gene of interest (GOI) is a fluorescence-Bcl-x_(L) fusion gene. Thefusion protein may include a fluorescence-Bcl-x_(L) construct (e.g.YFP-Bcl-x_(L) fusion or a Venus-Bcl-x_(L) fusion). In another preferredembodiment, the transgene is the YFP/Venus gene, not necessarily part ofa fusion protein. Venus is an enhanced yellow fluorescent protein (YFP)that is stable over a wide range of pH, folds quickly, and emits at30-fold the intensity of conventional YFP. Nagai T., Ibata K., Park E.S., Kubota M., Mikoshiba K. and Miyawaki A. (2002). A variant of yellowfluorescent protein with fast and efficient maturation forcell-biological applications. Nature Biotechnol, 20, 87-90.

In accordance to another embodiment, the system further comprises atleast one selective marker such as an antibiotic resistance marker. In apreferred embodiment, the GEE fusion protein and the at least onetransgene are introduced into the system on one vector and structurallyarranged to be expressed from one bidirectional promoter region andcomprising DNA binding sites in the vicinity of both promoters. In amore preferred embodiment, the GEE fusion protein and the transgene areintroduced in the system on separate vectors, each comprising aselective marker and the selective markers are not the same. Whenseparate vectors, both the GEE vector and the vector for the gene ofinterest (GOI) will contain selective markers. When the GEE isintroduced on a separate vector from the vector for the GOI, the GEEvector may be used to generate a stable algae cell line that will serveas the recipient for the second vector expressing the GOI. This stableGEE algae cell line will function to enhance the expression of thesecond vector containing the GOI.

In accordance to yet another embodiment, the algae compatibletranscriptional promoters are hsp70, rbcS, nitA, actin, tubA2 or acombination thereof.

In accordance to another yet embodiment, the GEE fusion proteincomprises a DNA binding domain functionally fused to an algae derivedp300 homologue having at least 80% identity over the HAT region to thep300 from Chlamydomonas reinhardtii. Preferably, the GEE fusion proteincomprises a DNA binding domain functionally fused to the HAT domain ofthe HAT region to the p300 from Chlamydomonas reinhardtii. It isnoteworthy that the p300 from mammalian species is much larger in sizeand is much less that 50% similar to Chlamydomonas reinhardtii p300.

The invention also provides a method of expressing a gene in algae athigher levels, comprising:

-   -   transforming algae with at least one vector comprising:        an algae compatible transcriptional promoter functionally        upstream of a coding sequence for a gene expression enhancer        (GEE) fusion protein, wherein the fusion protein comprises an        algae derived p300 functionally fused to the DNA binding        protein, wherein at least the portion of the coding sequence of        the DNA binding protein domain is codon optimized for improved        expression in an algae;    -   at least one transgene functionally downstream of an algae        compatible transcriptional promoter; and    -   at least one DNA region that is a binding site for the DNA        binding protein, in vicinity of at least one of said        transcriptional promoters;    -   selecting a transformed algae cell; and    -   detecting the expression of said GEE gene and/or said transgene        in algae.

In a preferred embodiment, the DNA Binding protein is the LexA bindingdomain, and more preferably the p300 is from Chlamydomonas reinhardtii.More preferably yet, the GEE fusion protein comprises the LexA bindingdomain functionally fused with the HAT domain of the p300 protein fromChlamydomonas reinhardtii.

In accordance to another embodiment, the transgene is a YFP-Bcl-x_(L)fusion protein or a Venus-Bcl-x_(L) fusion protein.

In accordance to yet another embodiment, the GEE fusion protein and saidtransgene are transformed in algae on separate vectors, first selectinga vector stably expressing the GEE fusion protein and then transformingthe selected algae with the vector comprising the transgene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the features of a vector in accordance to oneembodiment of the present invention. The direction of transcription isindicated by arrows. The figure indicates certain structural components,as discussed herein elsewhere. The linear drawing provides furtherdetails of the respective region of the vector: a fused LexA-p300protein coding region and a coding region of a GOI. In this embodiment,these two coding regions are transcribed in opposite directions (thusthe “bidirectional” nomenclature). These two coding regions areseparated by a locus comprising LexA binding sites.

FIGS. 2A and 2B illustrate another embodiment of the invention. FIG. 2Ais a vector expressing a LexA-p300 fusion protein. FIG. 2B illustrates avector expressing another gene which is advantageously introduced inalgae (“gene of interest or “GOI”). In accordance to this embodiment,each of the LexA-p300 fusion and the GOI have, at or near their 5′-ends,LexA binding site(s).

FIG. 3 compares the putative p300 protein from algae (Chlamydomonasreinhardtii (“Chlamy”) with known p300 proteins from the indicatedphylogenetically representative species. The lighter colored section ofeach bar represents the histone acetylase (HAT) domain. The HAT domainsare aligned for visualization purposes. These lighter bars includenumbers that are indicative of the percent identity of the HAT domain ofeach protein proteins within each panel, with the indicated percentagesof identity of each HAT protein to the p300 HAT domain of the p300protein from Chlamy. The figure is drawn to scale, both in respect tothe overall size of the p300 proteins and the location of the HAT domainwithin the protein.

DETAILED DESCRIPTION

Expression of transgenes in the algae is difficult. H. Cerutti, A.M.J.et al., The Plant Cell 9:925-945 (1997). Likewise, when the presentinventors transformed a microalgae with a construct expressing a yellowflorescence protein (“YFP”) fused to a cancer suppressing Bcl-x_(L) gene(the transcription driven by the rubisco promoter (rcbS2) and relying ona heat shock translational enhancer (HSP70)), the transformed microalgaefailed to produce fluorescence. However, transformants which survivedmarginally longer and were morphological affected (the result of limitedexpression of the Bcl-x_(L) gene) were observed. It is expected thatthat gene silencing contributed to the poor expression of the transgenesin algae.

The present invention provides an effective method to increase transgeneexpression in algae, preferably a green algae, more preferably amicroalgae. A preferred algae of the invention is an unicellular,photosynthetic algae. A yet more preferred algae is the microalgae. TheGOI transgene expressed in the algae in accordance to the invention isexpressed to a higher level. The expression is increased by at least50%, preferably about two to at least five fold, relative to theexpression of the same transgene engineered in the algae without thebenefit of the present invention. In respect of fluorescence transgenes,the expression is increased sufficiently to allow monitoring thefluorescence signal. More preferably, the fluorescence signal ismonitored in Chlamydomonas.

The transgene is introduced in algae. In accordance with an embodimentof the present invention, the transgene is placed on a vector. Thevector is a nucleic acid structure used to introduce a cassettecontaining a DNA sequence into an algae chromosome. The vector isintroduced in the nucleus of a host algae cell and the transgene istranscribed/translated in the algae. Methods of transformation of algaeare well known to artisans skilled in the art. For example, a vectorconstruct may be introduced via electroporation, via plasmidconjugation, and via particle bombardment. The transformed algae arerecovered on a solid nutrient media or in liquid media. Elizabeth HHarris, Chlamydomonas As A Model Organism, Annual Review of PlantPhysiology and Plant Molecular Biology 52:363-406 (2001) and EMBOPractical Course: Molecular Genetics of Chlamydomonas, Laboratoryprotocols. Geneva, Sep. 18-28, 2006.

A preferred vector of the invention is a plasmid capable of integratingthe DNA sequence of interest into a chromosome of the algae. There are alarge numbers of vectors known and characterized. A preferred vector ofthe invention is pSP124. Lumbreras et al., Efficient foreign geneexpression in Chlamydomonas reinhardtii mediated by an endogenousintrons, The Plant Journal 14(4):441-447 (1998).

Methods of engineering vectors are well known in the art. The vectorbackbone may include genes encoding transformation markers, to indicatetransformation of the host cell with the vector. A transformation markermay be a selective marker gene used to select cells in which the vectoris present from normal cells without the vector. Selective markers arewell known to artisans skilled in the art. Commonly used selectivemarkers include genes that confer resistance to specific antibioticssuch as bleomycin. Only cells containing the vector grow in mediacontaining the antibiotic. Other vector backbones may also includemarker genes that merely indicate which cells were transformed. Whensuch markers are used, cells with and without the vector will grow butthe cells containing the vector can be distinguished from those nothaving the vector because they display a specific characteristicconferred by the vector, e.g., color. A commonly used transformationmarker gene is the yellow or green fluorescence gene. Cells containing avector with such a gene will be yellow or green. Other commontransformation markers include various luciferase genes. Cellscontaining the luciferase genes emit light.

Any effective combination of gene expression regulatory featurescompatible with expression of genes in the algae nucleus can beincorporated in the vector. The plasmid may include different types ofpromoters, for example constitutive promoters or inducible promoters.Preferred transcriptional promoters in accordance to the inventioninclude the hsp70 (“heat shock protein” promoter), rbcS (“rubisco smallsubunit” promoter) and tubA2 (“actin” promoter). The vector employssuitable translational enhancer elements, generally referred to as5′untranslated regions or “5′UTR.” Preferred enhancers in accordance tothe invention are the tubA2 intron 1, the HSP70 enhancer, and the rcbS2intron 1. The vector of the invention includes also effectivetranslational terminators, 3′UTR. Examples of preferred 3′-UTR sequencesinclude the tubA2, HSP70, and rcbS2 3′UTR. Other effective promoters,transcription enhancers and terminators may, in particular combinations,may produce satisfactorily high and stable expression.

Some of these options are illustrated in FIGS. 1 and 2. The featuresselected to be exemplified in FIGS. 1 and 2 include the promoter and3′UTR regions of the Chlamy genes: tubA2 encoding actin (Tubulin); rbcS2encoding the rubisco small subunit; or nitA encoding nitrate reductase.Furthermore, the hsp70A/rbcS2 tandem promoter is a preferred driver oftransgene expression. Schroda M., Beck C. F. and Vallon A., Sequenceelements within an hsp70 promoter counteract transcriptional transgenesilencing in Chlamydomonas. Plant J. 31:445-455 (2002). This chimericpromoter contains the enhancer region of thenucleo-cytoplasmic-localized 70 kD heat shock protein gene (NCBI GenBankID: M76725; by 572-833) and the promoter from the nuclear rubisco smallsubunit gene (NBCI GenBank ID: X04472; bp 934-1142). Additionally, thefirst intron (bp 1307-1451) and 3′-untranslated region (bp 2401-2632) ofthe rbcS2 gene may be included to further promote stable transgeneexpression.

In accordance with an embodiment of the present invention, one or morevectors are used to introduce a cassette that contains a gene ofinterest (“GOI”) and a gene silencing inhibitor into the nucleus DNA ofalgae, e.g., a Chlamy nucleus. The GOI can be any gene desirablyexpressed in algae. Viable genes of interest include genes involved incontrolling algae's metabolic pathways. For example, in one embodimentof the present invention the Bcl-x_(L) gene can be inserted andexpressed in the algae's nucleus. Bcl-x_(L) is an abbreviation forB-cell lymphoma extra-large; it is known to be an inhibitor of apoptosis(programmed cell death). Boise L. H. et al., Bcl-x, a bcl-2-related Genethat Functions as a Dominant Regulator of Apoptotic Cell Death, Cell74:597-608 (1993). In another embodiment genes affecting lipid orisoprenoid production pathways are desirably introduced. Due toBcl-x_(L)'s ability to inhibit apoptosis, its expression allows algaecells to live longer. A longer lifespan for microalgae enables the useof microalgae in various industrial applications such asphotobioreactors.

A gene silencing inhibitor is also introduced into the algae. A genesilencing inhibitor is a peptide that induces relaxation of nucleosomesin the algae's nucleus. Gene silencing inhibitors include histone acetyltransferases (HATs) and other peptides that modify elements of thenucleosome, causing the chromatin structure to relax and to allowtranscription factors to access the gene of interest. HAT proteins andthe HAT domains of p300 and of other HAT proteins are known to causehistone acetylation and can be utilized in the invention. In accordanceto the invention the domain responsible for the acetylation activity orthe whole protein is deployed. See Fukuda H, et al., Brief Funct.Genomic Proteomic, 5(3):190-208 (2006); Renthal W. and Nestler E. J.,Semin Cell Dev Biol. 20(4):387-94 (Epub 2009); and Lin Y. Y. et al.,Genes Dev., 22(15):2062-74 (2008).

One preferred embodiment of the present invention utilizes a p300protein as a gene silencing inhibitor. More preferably, a Chlamy derivedp300 protein is utilized. In a yet more preferred embodiment, the Chlamyp300 protein is the homologue detailed in FIG. 3. In a further morepreferred embodiment, only the HAT domain of the Chlamy p300 gene isutilized. See FIG. 3 and relevant portion of SEQ ID NO 4.

FIG. 3 shows an alignment comparison of the Chlamy p300 withphylogenetically distinct other p300 homologues. The lighter coloredsection of each bar represents the histone acetylase (HAT) domain. TheHAT domains are aligned for visualization purposes. These lighter barsinclude numbers that are indicative of the percent identity of the HATdomain of each protein proteins with the indicated percentages ofidentity of each HAT protein to the p300 HAT domain of the p300 proteinfrom Chlamy. FIG. 3 is drawn to scale, both in respect to the overallsize of the p300 proteins and the location of the HAT domain within theprotein.

Table 1, exemplifies the highly conserved nature of the p300 proteinsand particularly conserved nature of the HAT domains.

TABLE 1 Comparison of HAT domain identity within each phylogeneticclade.

 - 100%

 - 100%

 - 100%

 - 100% V. carteri - 85% G. max - 91% A. gambiae - 92% M. mulatta - 100%O. sativa - 91% C. floridanus - 89% O. cuniculus - 100% S. bicolor - 90%R. norvegicus - 99% P. trichocarpa - 88% H. musculus - 99% MicroalgaeHigher Plants Insects Mammals The bolded organism at the top of eachcolumn is the representative species to which all other percentidentities are based.

Indeed, the percent identity between plant and mammalian p300 homologuesis also very high, typically at least about 80%. See US PatentPublication US2003/0145349. However, the homology of the Chlamy p300homologue to other organisms is lower. Likewise, the p300 full proteinof Chlamydomonas reinhardtii is 11.5% identical and further 9.9% similarto the mouse p300 protein; 9.1% identical and a further 4.7% similar tothe Drosophila p300 protein; and 23.6% identical and a further 9.9%similar to the Arabidopsis p300 protein. The Chlamy derived protein hasN-terminal or C-terminal regions which are shorter and dissimilar intheir location visa-vie the HAT domain to these of the mammalian orplant p300 proteins. See FIG. 3. This is suggestive of proteins withoverall distinct functions and phylogeny.

The various proteins p300 homologues in FIG. 1 and described hereinelsewhere are:

-   C. reinhardtii p300/HAT Protein ID: 159467703 from NCBI Database.-   V. carteri p300/CBP Protein ID: 300256266 from NCBI Database.-   S. bicolor putative p300 Protein ID: C5XTZ4 from Universal Protein    Resource.-   P. trichocarpa GenBank ID: POPTR_(—)007s15090 from Joint Genome    Institute Database.-   G. max Protein ID: PF02135 from Joint Genome Institute Database.-   A. thaliana HAC1/p300/CBP GenBank ID: NM_(—)106550.3 from NCBI    Database.-   O. saliva p300/CBP Protein ID: 108792657 from NCBI Database.-   D. melanogaster CBP/HAT Genbank ID: NM_(—)079903.2 from NCBI    Database.-   A. gambiae HAT Protein ID: 158289391 from NCBI Database.-   C. floridanus CBP Protein ID: 307172990 from NCBI Database.-   M. musculus E1A/BP/p300 GenBank ID: NM_(—)177821.6 from NCBI    Database.-   O. cuniculus p300 Protein ID: 291410334 from NCBI Database.-   R. norvegicus p300 Protein ID: XP_(—)576312.3 from NCBI Database.-   M. mulatta p300 HAT Protein ID: XP_(—)001102844.1 from NCBI    Database.-   H. sapiens p300 Protein ID: NP_(—)001420.2 from NCBI Database.

In another preferred embodiment of the present invention, the genesilencing inhibitor is functionally tethered or, preferably, fused to aDNA binding protein or domain thereof (the tethered/fused protein orits/their gene hereinafter are referred to as the gene expressionenhancer unit, or “GEE”). The DNA binding protein or domain binds to aparticular DNA sequence (Binding Site or “BS”), bringing the genesilencing inhibitor to its histone target at a location in the vicinityof the BS and thereby inducing relaxation of the nucleosome at thatgenetic location. As the nucleosome relaxes, the nearby DNA sequence isexposed to transcription factors and is more actively transcribed.

In accordance to a preferred embodiment, the invention requires theexpression in an algae protein that binds specific DNA sequences, whichsequences can be engineered upstream of any GOI for expression in algae.The DNA binding protein/domain can be any protein having known DNAbinding sites can be used. Examples of proteins targeting specific DNAmotifs applicable to this invention include the Gal4 protein and EarlyGrowth Response Protein 1. DNA binding site motifs for these proteinsare known. Likewise, the binding domains of these as well as the LexAprotein are known and are preferentially used, instead of thefull-length protein. See for example Young, K., Biol. Reprod.,58:302-311 (1998) and Joung, J. et al., Proc. Natnl. Acad. Sci.,97:7382-7 (2000). The DNA binding site (BS) for Gal4 is5′-CGGAGGACAGTCCTCCG-3′ (SEQ ID NO 10).

LexA is a preferred example of a DNA binding protein. LexA is a gene ofbacterial origin. LexA proteins or genes are not known in algae. Thus,it is unlikely that the Chlamy genome will contain the DNA bindingsequence of LexA. The function of LexA in the context of the inventionis to bind a particular DNA sequence (binding site, “BS”). LexA bindingsites are found upstream promoters in a number of microorganisms. Aconsensus BS sequence for LexA is CTGTATATATATACAG. SEQ ID NO 9. Thebinding domain of the LexA protein is known and, for the purpose of theinvention, it is preferred to employ only the binding domain. ProteinID: 2293118 from NCBI Database: MKALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEEHLKALARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAAGEPLLAQQHIEGHYQVDPSLFKPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARIDDEVTVKRLKKQGNKVELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGDWLEFPGIRRPWRPLESTCSQANSGRISYDL (SEQ ID NO 11).

As noted above, the DNA binding protein or domain thereof, preferablythe LexA domain, is constructed to translate in a protein allowing theDNA binding domain and a nucleosome relaxation protein to act inconcert. Any nucleosome relaxation protein might be used. Preferably, asnoted above, a Chlamy p300 domain is used.

Without being limited to a single mechanism of action, it is proposedthat one partner binds to the DNA, the other acetylates nearby histones,thereby creating a looser association between the DNA and the histonesat that site. Therefore any method to render the DNA binding domain andthe acetylase domain spatially close to each other is preferred. A fusedprotein is more preferred. The order of the two units (N-terminalproximity) within the fusion protein is not critical. However, in thep300-LexA binding domain example, it is preferred that LexA bindingdomain is at the N-terminal end of the fusion. “Functional” fusionproteins are designed. By way of example, certain linker regions areintroduced to allow flexibility, orientation or simply “dead” proteinsequence corresponding to strategically placed genetic engineeringfeatures such as primers and restriction enzyme sites.

Preferably, the GEE can be a p300 peptide homolog and the DNA bindingdomain can be LexA binding domain, creating a p300-LexA binding domainfusion protein and its gene construct. Preferably, that fusion is analgae p300-LexA binding domain fusion. More preferably, the fusion isthe Chlamy p300-LexA fusion. Alternatively, the fusion comprises selectdomains of the Chlamy p300-LexA proteins. See SEQ ID NO 4. Yet morepreferably, the fusion, at the nucleic acid level, comprises a LexAsequence modified in its codon usage for higher yield when expressed inalgae. Preferably, the whole of the GEE fusion protein gene was designedfor preferred codon usage in algae, even if part of the gene (p300) isan algae derived gene, as provided by SEQ ID NO 1 and SEQ ID NO 3.Indeed, the transgene (GOI) and other genes in the system preferably arecodon optimized based on codon frequency in algae.

It should be noted that other algae p300 homologues or theiracetylasehistone acetyltransferase (HAT) domains may be preferentiallyused in the invention. However, these preferred homologues must be atleast about 60% identical to the Chlamy p300, preferably at least about70% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical or more. A p300 homologue fromV. carteri (algae) was recently identified. It has about 85% identity tothe Chlamy p300, over the HAT domains.

The LexA-p300 fusion DNA (SEQ ID 1) is the nucleotide sequence encodinga fusion protein (exemplary GEE) comprising the LexA binding domain andthe full length Chlamy p300 sequence, all of the fusion designed toreflect preferred codon usage in algae. It was adapted to the nuclearcodon usage of C. reinhardtii according to the table provided by theKazusa Codon Usage Database (Species ID: 3055), using Gene Designersoftware from DNA 20. The sequence up to nucleotide 690 is that of theLexA DNA binding domain and the fell length C. reinhardtii p300 sequencebegins at nucleotide 700. A 3-amino acid peptide linker (GVL) connectsLexA binding domain and p300, which represents the DNA restriction sitePpuMI (9 bp). The LexA gene sequence is codon-optimized for C.reinhardtii nuclear expression using AA sequence from Protein ID:2293118 from NCBI Database:

SEQ ID NO 1 1 ATGAAGGCTCTGACCGCTCGCCAGCAGGAGGTGTTTGATCTGATTCGGGA 51CCATATCAGCCAAACGGGCATGCCCCCTACGCGCGCGGAGATCGCGCAAC 101GGCTGGGCTTCCGCTCCCCGAACGCGGCTGAGGAGCACCTGAAGGCGCTG 151GCGCGCAAGGGTGTGATTGAGATCGTCTCCGGCGCGTCGCGGGGCATTCG 201GCTGCTGCAGGAGGAGGAGGAGGGTCTGCCTCTGGTGGGGCGGGTGGCTG 251CGGGCGAGCCCCTGCTGGCCCAGCAGCACATTGAGGGCCACTACCAAGTG 301GACCCGTCCCTCTTCAAGCCGAACGCCGATTTCCTGCTGCGCGTCAGCGG 351TATGAGCATGAAGGACATCGGCATCATGGACGGTGACCTGCTGGCCGTGC 401ATAAGACGCAGGACGTGCGCAACGGCCAAGTGGTCGTCGCCCGCATCGAT 451GACGAGGTGACCGTGAAGCGCCTGAAGAAGCAGGGGAACAAGGTCGAGCT 501GCTGCCCGAGAACAGCGAGTTCAAGCCCATCGTGGTGGATCTGCGCCAGC 551AATCCTTCACCATCGAGGGCCTGGCGGTGGGCGTGATCCGCAACGGCGAC 601TGGCTGGAGTTCCCGGGCATCCGCCGCCCGTGGCGCCCTCTGGAGTCCAC 651GTGCTCGCAGGCCAACTCCGGCCGCATTAGCTACGACCTGGGGGTCCTTA 701TGGTGCCGATGGGCGCGCCCGCTATGCCCATGGGCAACAACGGCTCGCCC 751ATGCTGAACGGCATGGGTATGTTCAACGCCCCGCAGCAGACCGTGCCCAA 801CGGCGGGCCGGGTGGCGTGAACCCCATGGGCCAGGTGCCGGCGATGCCTG 851CGCCGATCCCCAACGGCGGTCTGCCCGGTATGAACGCTGCCGGCGGTGCC 901GACGATCCTGCGAAGCAGCGGGAGCAATCGATCCAGAAGCAGCAGCGCTG 951GCTGCTGTTCCTGCGGCACTGCGCGAAGTGCCGGGCTCCCGGCGAGGACT 1001GCCAGCTGAAGTCCCAGTGCAAGTTCGGCAAGCAGCTGTGGCAGCACATC 1051CTGTCGTGCCAAAACCCGGCCTGCGAGTACCCGCGCTGCACCAACTCCAA 1101GGATCTGCTCAAGCACCACCAGAAGTGCCAGGAGCAGACCTGCCCCGTGT 1151GCATGCCGGTGAAGGACTACGTGAAGAAGACGCGCCAGGCGACCCAACAG 1201CAGCAACAAATGCAGCAACAACAGCAAATCCAGCAACAGCAACAACAACA 1251AATGCAACAGCAACAGATGCAACAGCAGCAGCTCCAGCAGCAGCAGATGC 1301AACAACAACAGCAGATGCAGCAGCAGCAACAGCCCGGCGTGGGCGCCAAC 1351TTCATGCCCACCCCGCCCATGATGCCGAACGGCATGTTCCCTCAACAGCA 1401GCCCCAGCAGGCGATGCGCCTGAACGCCAACGGCCTCGGCGGCCAGAAGC 1451GCCCCCACGAGATGATGGGTATGTCCAGCGGCGGCATGGACGGTATGAAC 1501CAGATGGTGCCCGTCGGCGGCGGCGGCATGGGCATGTCGATGCCGATGGG 1551TATGAACAACCCCATGCAGGGCGGTATGCCCCTGCAGCCTCCGCCCCAGG 1601TGCAGGCTCCCGGTCAGGGCCCCATGATGAGCGCCCCTCAGCAGCAACAG 1651CAGCAACCGGCCCCTAAGCGGGCGAAGACCGACGATGTGCTGCGCCAGAA 1701CACGGGCACCAGCCTCCTGGAGACGTTCGACGCCAAGCAGATCCGCGTGC 1751ACGTGGACCTGATCCGCGCTGCCGCGGTGACCCAGAAGGCCCAGCAGCCT 1801CCCCCGGCTAACCCCGACGACGCGTGCAAGGTCTGCGCGCTGACGAAGCT 1851GTCGTTCGAGCCCCCGGTGATTTACTGCTCGAGCTGCGGCCTGCGCATCA 1901AGCGCGGCCAGATCTTCTACAGCACGCCTCCGGACCACGGCAACGACCTG 1951AAGGGTTACTTCTGCCACCAGTGCTTCACCGACCAGAAGGGCGAGCGCAT 2001CCTGGTGGAGGGCGTCTCGATCAAGAAGAGCGACCTGGTGAAGCGCAAGA 2051ACGATGAGGAGATCGAGGAGGGGTGGGTGCAGTGCGACCACTGCGAGGGC 2101TGGGTGCACCAGATTTGCGGCATGTTCAACAAGGGCCGGAACAACACGGA 2151CGTCCACTACCTGTGCCCTGACTGCCTGGCCGTGGGCTACGAGCGCGGCC 2201AGCGCCAGAAGACGGAGGTCCGCCCCCAGGCGATGCTCGAGGCGAAGGAT 2251CTGCCCACGTCCCGGCTGTCCGAGTTTATTACGGAGCGCCTGAACCGCGA 2301GCTGGAGAAGGAGCACCACAAGCGGGCTGAGCAGCAGGGCAAGCCGCTGC 2351ACGAGGTGGCGAAGCCCGAGCCCCTGACCGTGCGGATGATCAACTCCGTG 2401ATGAAGAAGTGCGAGGTCAAGCCGCGCTTCCACGAGACGTTCGGCCCCAC 2451CGACGGCTACCCCGGGGAGTTCGGCTACCGGCAGAAGGTGCTGCTGCTGT 2501TCCAAAGCCTGGACGGTGTCGACGTGTGCCTGTTCTGCATGTACGTGCAG 2551GAGTACGGCAAGGACTGCCCTGCGCCCAACACCAACGTGGTGTACCTGTC 2601GTATCTGGACTCCGTCAAGTACTTCCGCCCTGAGATTCCCTCGGCCCTGG 2651GCCCTGCCGTGTCGCTGCGCACCTTCGTGTACCACCAACTCCTGATCGCC 2701TACGTGGAGTTTACCCGCAACATGGGTTTTGAGCAGATGTACATTTGGGC 2751GTGCCCGCCGATGCAAGGCGACGACTACATCCTGTACTGCCACCCGACCA 2801AGCAGAAGACGCCGCGCTCGGACCGCCTGCGCATGTGGTACATTGAGATG 2851CTGAAGCTGGCGAAGGAGGAGGGTATCGTGAAGCACCTGAGCACGCTGTG 2901GGATACGTACTTCGAGGGCGGTCGCGACCACCGGATGGAGCGCTGCTCGG 2951TCACGTACATTCCGTACATGGAGGGCGACTACTGGCCCGGCGAGGCTGAG 3001AACCAGCTCATGGCCATTAACGACGCGGCCAAGGGCAAGCCTGGGACCAA 3051GGGTGCGGGCAGCGCCCCGAGCCGCAAGGCCGGTGCCAAGGGCAAGCGCT 3101ACGGCGGTGGCCCCGCCACGGCTGATGAGCAGCTGATGGCCCGCCTCGGT 3151GAGATCCTGGGCGGGAACATGCGGGAGGACTTCATTGTGGTCCACATGCA 3201GGTGCCCTGCACGTTCTGCCGCGCTCACATTCGGGGTCCGAACGTGGTGT 3251ACCGCTATCGGACGCCGCCTGGCGCGACCCCTCCCAAGGCTGCCCCCGAG 3301CGCAAGTTCGAGGGCATCAAGCTGGAGGGCGGTGGCCCCAGCGTGCCCGT 3351GGGCACCGTCTCGAGCCTGACGATCTGCGAGGCGTGCTTTCGCGACGAGG 3401AGACGCGCACGCTGACCGGCCAACAGCTGCGCCTGCCCGCTGGCGTGTCG 3451ACCGCTGAGCTCGCGATGGAGAAGCTGGAGGAGATGATCCAGTGGGACCG 3501CGACCCTGACGGCGACATGGAGAGCGAGTTCTTCGAGACGCGGCAGACCT 3551TCCTGTCGCTGTGCCAGGGCAACCACTACCAGTTCGACACCCTCCGCCGC 3601GCTAAGCACTCGTCGATGATGGTGCTCTACCACCTGCACAACCCCCACTC 3651GCCGGCGTTCGCGTCCTCGTGCAACCAGTGCAACGCCGAGATCGAGCCGG 3701GCAGCGGCTTTCGCTGCACCGTGTGCCCCGACTTCGACATGTGCGCCAGC 3751TGCAAGGTCAACCCTCATAAGCGCGCCCTGGACGAGACGCGCCAGCGGCT 3801GACCGAGGCCGAGCGCCGGGAGCGCAACGAGCAGCTGCAGAAGACCCTCG 3851CCCTGCTGGTGCACGCCTGCGGCTGCCACAACAGCGCGTGCGGCTCCAAC 3901AGCTGCCGCAAGGTGAAGCAGCTGTTCCAGCACGCGGTCCACTGCCAGAG 3951CAAGGTGACCGGGGGCTGCCAGCTGTGCAAGAAGATGTGGTGCCTGCTGA 4001ACCTGCACGCCAAGTCCTGCACCCGCGCGGACTGCCCGGTGCCGCGCTGC 4051AAGGAGCTGAAGGAGCTGCGCCGGCGCCAAACGAACCGGCAGGAGGAGAA 4101GCGCCGGGCGGCCTACGCCGCTATGCTGCGCAACCAGATGGCCGGCAGCC 4151AGGCTCCGCGCCCCATGTAA.

LexA-p300 Fusion Protein (SEQ ID NO 2) is the respective proteinsequence encoded by the nucleic acid sequence of SEQ ID NO 1. The LexAbinding domain is the sequence up to and including amino acid 230 andthe full-length p300 HAT domain sequence begins at amino acid 234. A3-amino acid peptide linker (GVL) connects LexA binding domain and p300,which represents the DNA restriction site PpuMI (9 bp):

SEQ ID NO 2 1 MKALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEEHLKAL 51ARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAAGEPLLAQQHIEGHYQV 101DPSLFKPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARID 151DEVTVKRLKKQGNKVELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGD 201WLEFPGIRRPWRPLESTCSQANSGRISYDLGVLMVPMGAPAMPMGNNGSP 251MLNGMGMFNAPQQTVPNGGPGGVNPMGQVPAMPAPIPNGGLPGMNAAGGA 301DDPAKQREQSIQKQQRWLLFLRHCAKCRAPGEDCQLKSQCKFGKQLWQHI 351LSCQNPACEYPRCTNSKDLLKHHQKCQEQTCPVCMPVKDYVKKTRQATQQ 401QQQMQQQQQIQQQQQQQMQQQQMQQQQLQQQQMQQQQQMQQQQQPGVGAN 451FMPTPPMMPNGMFPQQQPQQAMRLNANGLGGQKRPHEMMGMSSGGMDGMN 501QMVPVGGGGMGMSMPMGMNNPMQGGMPLQPPPQVQAPGQGPMMSAPQQQQ 551QQPAPKRAKTDDVLRQNTGTSLLETFDAKQIRVHVDLIRAAAVTQKAQQP 601PPANPDDACKVCALTKLSFEPPVIYCSSCGLRIKRGQIFYSTPPDHGNDL 651KGYFCHQCFTDQKGERILVEGVSIKKSDLVKRKNDEEIEEGWVQCDHCEG 701WVHQICGMFNKGRNNTDVHYLCPDCLAVGYERGQRQKTEVRPQAMLEAKD 751LPTSRLSEFITERLNRELEKEHHKRAEQQGKPLHEVAKPEPLTVRMINSV 801MKKCEVKPRFHETFGPTDGYPGEFGYRQKVLLLFQSLDGVDVCLFCMYVQ 851EYGKDCPAPNTNVVYLSYLDSVKYFRPEIPSALGPAVSLRTFVYHQLLIA 901YVEFTRNMGFEQMYIWACPPMQGDDYILYCHPTKQKTPRSDRLRMWYIEM 951LKLAKEEGIVKHLSTLWDTYFEGGRDHRMERCSVTYIPYMEGDYWPGEAE 1001NQLMAINDAAKGKPGTKGAGSAPSRKAGAKGKRYGGGPATADEQLMARLG 1051EILGGNMREDFIVVHMQVPCTFCRAHIRGPNVVYRYRTPPGATPPKAAPE 1101RKFEGIKLEGGGPSVPVGTVSSLTICEACFRDEETRTLTGQQLRLPAGVS 1151TAELAMEKLEEMIQWDRDPDGDMESEFFETRQTFLSLCQGNHYQFDTLRR 1201AKHSSMMVLYHLHNPHSPAFASSCNQCNAEIEPGSGFRCTVCPDFDMCAS 1251CKVNPHKRALDETRQRLTEAERRERNEQLQKTLALLVHACGCHNSACGSN 1301SCRKVKQLFQHAVHCQSKVTGGCQLCKKMWCLLNLHAKSCTRADCPVPRC 1351KELKELRRRQTNRQEEKRRAAYAAMLRNQMAGSQAPRPM*.

LexA-p300 HAT domain DNA (SEQ ID NO 3) is a nucleic acid sequencecorresponding to a gene encoding the LexA bindingdomain-acetyl-transferase (HAT) domain of the Chlamy p300 protein.Similarly, the LexA binding domain is the sequence up to and includingnucleotide 690 and the p300 HAT domain sequence begins at nucleotide700. A 3-amino acid peptide linker (GVL) connects LexA binding domainand p300, which represents the DNA restriction site PpuMI (9 bp).

SEQ ID NO 3 1 ATGAAGGCTCTCACCGCTCGCCAACAGGAGGTCTTTGATCTGATTCGCGA 51CCACATCTCGCAGACCGGCATGCCGCCGACCCGGGCGGAGATTGCTCAGC 101GGCTGGGCTTCCGGAGCCCCAACGCGGCCGAGGAGCACCTGAAGGCCCTC 151GCGCGCAAGGGGGTGATCGAGATTGTCTCCGGCGCTAGCCGCGGCATCCG 201CCTGCTGCAGGAGGAGGAGGAGGGCCTGCCGCTGGTCGGGCGGGTCGCGG 251CCGGGGAGCCTCTGCTGGCCCAGCAGCACATCGAGGGCCACTACCAAGTG 301GACCCCTCGCTGTTTAAGCCCAACGCGGACTTCCTGCTCCGGGTGTCGGG 351CATGAGCATGAAGGACATCGGCATCATGGACGGCGACCTCCTGGCGGTGC 401ACAAGACCCAGGACGTGCGCAACGGCCAGGTGGTCGTCGCGCGGATTGAC 451GACGAGGTGACCGTGAAGCGGCTGAAGAAGCAGGGCAACAAGGTCGAGCT 501GCTGCCCGAGAACTCGGAGTTCAAGCCTATCGTGGTCGACCTGCGCCAGC 551AGTCCTTCACCATCGAGGGCCTGGCCGTGGGGGTCATCCGCAACGGTGAC 601TGGCTGGAGTTCCCCGGCATCCGGCGCCCGTGGCGGCCGCTGGAGTCCAC 651CTGCAGCCAGGCGAACTCCGGCCGCATCTCCTACGATCTGGGGGTCCTTG 701AGGTGGCCAAGCCGGAGCCGCTGACCGTGCGGATGATCAACAGCGTGATG 751AAGAAGTGCGAGGTCAAGCCCCGCTTCCACGAGACGTTCGGTCCGACCGA 801CGGTTACCCCGGGGAGTTCGGCTACCGGCAGAAGGTGCTCCTCCTGTTCC 851AGTCCCTCGACGGCGTCGACGTGTGCCTGTTCTGCATGTACGTGCAGGAG 901TACGGGAAGGACTGCCCGGCGCCCAACACGAACGTGGTGTACCTGAGCTA 951CCTGGACTCCGTCAAGTATTTCCGCCCCGAGATTCCCAGCGCCCTGGGCC 1001CTGCGGTGAGCCTGCGGACCTTCGTGTACCACCAGCTCCTGATTGCGTAC 1051GTGGAGTTCACGCGCAACATGGGCTTCGAGCAGATGTACATTTGGGCGTG 1101CCCCCCCATGCAGGGGGACGACTATATCCTGTATTGCCATCCCACGAAGC 1151AGAAGACCCCGCGCTCGGACCGCCTGCGCATGTGGTACATCGAGATGCTG 1201AAGCTGGCTAAGGAGGAGGGCATCGTGAAGCACCTGTCGACGCTGTGGGA 1251CACCTACTTCGAGGGCGGTCGCGACCACCGGATGGAGCGCTGCAGCGTGA 1301CCTACATCCCCTACATGGAGGGCGACTACTGGCCTGGCGAGGCCGAGTAA.

LexA-p300 HAT domain AA (SEQ ID NO 4) is an exemplary GEE proteinsequence of a LexA binding domain-Chlamy p300 protein, where the Chlamyp300 is limited to the histone acetyl-transferase (HAT) domain of theChlamy p300 enzyme. The LexA binding domain is the sequence up to andincluding amino acid 230 and the p300 HAT domain sequence begins atamino acid 234. The 3-amino acid peptide linker (GVL) connects LexAbinding domain and p300:

SEQ ID NO 4 1 MKALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEEHLKAL 51ARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAAGEPLLAQQHIEGHYQV 101DPSLFKPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARID 151DEVTVKRLKKQGNKVELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGD 201WLEFPGIRRPWRPLESTCSQANSGRISYDLGVLEVAKPEPLTVRMINSVM 251KKCEVKPRFHETFGPTDGYPGEFGYRQKVLLLFQSLDGVDVCLFCMYVQE 301YGKDCPAPNTNVVYLSYLDSVKYFRPEIPSALGPAVSLRTFVYHQLLIAY 351VEFTRNMGFEQMYIWACPPMQGDDYILYCHPTKQKTPRSDRLRMWYIEML 401KLAKEEGIVKHLSTLWDTYFEGGRDHRMERCSVTYIPYMEGDYWPGEAE*.

Codon-optimized Venus gene sequence is a preferred embodiment:

SEQ ID NO 5 1 ATGGTGTCGAAGGGTGAGGAGCTGTTTACCGGTGTCGTGCCTATTCTGGT 51GGAGCTCGACGGCGACGTCAACGGGCACAAGTTTTCGGTGTCCGGCGAGG 101GTGAGGGGGACGCGACGTACGGCAAGCTCACGCTGAAGCTGATCTGCACC 151ACCGGCAAGCTGCCCGTCCCCTGGCCGACGCTGGTGACCACCCTGGGCTA 201CGGCCTGCAGTGCTTCGCCCGCTACCCGGACCACATGAAGCAGCACGACT 251TCTTCAAGTCGGCCATGCCCGAGGGGTACGTGCAGGAGCGCACGATCTTC 301TTTAAGGACGATGGCAACTACAAGACCCGCGCTGAGGTGAAGTTCGAGGG 351CGATACGCTGGTGAACCGCATCGAGCTCAAGGGCATCGACTTCAAGGAGG 401ACGGCAACATCCTGGGTCACAAGCTGGAGTACAACTACAACTCCCACAAC 451GTGTACATCACGGCGGATAAGCAGAAGAACGGCATCAAGGCCAACTTTAA 501GATTCGCCATAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACC 551AGCAGAACACCCCGATCGGCGACGGCCCCGTGCTGCTGCCCGATAACCAC 601TACCTCAGCTACCAGTCGGCCCTGTCCAAGGATCCCAACGAGAAGCGCGA 651TCACATGGTCCTCCTGGAGTTCGTGACCGCCGCTGGCATCACCCTGGGCA 701TGGACGAGCTGTACAAGTAA.

SEQ ID NO 6 is the protein encoded by the nucleic acid of SEQ ID NO 5.The Venus AA sequence:

SEQ ID NO 6 1 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICT 51TGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIF 101FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN 151VYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH 201YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*.

SEQ ID NO 7 is a nucleic acid encoding a Venus-Bcl-x_(L) fusion of theinvention. It was designed to represent preferred codon usage in algae.The sequence up to and including nucleotide 717 represents Venus. A3-amino acid peptide linker (GVL) connects Venus and Bcl-x_(L), whichrepresents the DNA restriction site PpuMI (9 bp). Bcl-x_(L) begins atnucleotide 726.

SEQ ID NO 7 1 ATGGTGTCGAAGGGTGAGGAGCTGTTTACCGGTGTCGTGCCTATTCTGGT 51GGAGCTCGACGGCGACGTCAACGGGCACAAGTTTTCGGTGTCCGGCGAGG 101GTGAGGGGGACGCGACGTACGGCAAGCTCACGCTGAAGCTGATCTGCACC 151ACCGGCAAGCTGCCCGTCCCCTGGCCGACGCTGGTGACCACCCTGGGCTA 201CGGCCTGCAGTGCTTCGCCCGCTACCCGGACCACATGAAGCAGCACGACT 251TCTTCAAGTCGGCCATGCCCGAGGGGTACGTGCAGGAGCGCACGATCTTC 301TTTAAGGACGATGGCAACTACAAGACCCGCGCTGAGGTGAAGTTCGAGGG 351CGATACGCTGGTGAACCGCATCGAGCTCAAGGGCATCGACTTCAAGGAGG 401ACGGCAACATCCTGGGTCACAAGCTGGAGTACAACTACAACTCCCACAAC 451GTGTACATCACGGCGGATAAGCAGAAGAACGGCATCAAGGCCAACTTTAA 501GATTCGCCATAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACC 551AGCAGAACACCCCGATCGGCGACGGCCCCGTGCTGCTGCCCGATAACCAC 601TACCTCAGCTACCAGTCGGCCCTGTCCAAGGATCCCAACGAGAAGCGCGA 651TCACATGGTCCTCCTGGAGTTCGTGACCGCCGCTGGCATCACCCTGGGCA 701TGGACGAGCTGTACAAGGGGGTCCTTATGAGCCAGAGCAACCGGGAGCTG 751GTGGTGGACTTCCTGAGCTACAAGCTGAGCCAAAAGGGCTATAGCTGGTC 801GCAGTTCTCCGACGTCGAGGAGAACCGGACCGAGGCCCCCGAGGGGACCG 851AGTCCGAGATGGAGACGCCGAGCGCGATTAACGGCAACCCGAGCTGGCAC 901CTGGCGGACTCCCCTGCCGTGAACGGCGCGACCGGCCACAGCTCCAGCCT 951GGACGCGCGCGAGGTCATCCCGATGGCGGCCGTGAAGCAGGCCCTCCGCG 1001AGGCCGGCGACGAGTTCGAGCTGCGCTATCGCCGCGCTTTCTCGGACCTG 1051ACCAGCCAGCTGCACATCACCCCCGGCACGGCTTACCAAAGCTTCGAGCA 1101GGTGGTGAACGAGCTGTTCCGCGACGGCGTGAACTGGGGTCGCATCGTGG 1151CGTTCTTCAGCTTCGGCGGTGCGCTGTGCGTGGAGAGCGTCGACAAGGAG 1201ATGCAGGTGCTGGTGTCGCGCATTGCGGCTTGGATGGCCACCTACCTGAA 1251CGACCACCTGGAGCCCTGGATTCAGGAGAACGGCGGCTGGGACACCTTCG 1301TCGAGCTGTACGGCAACAACGCTGCGGCGGAGAGCCGCAAGGGCCAAGAG 1351CGGTTCAACCGCTGGTTCCTCACGGGGATGACCGTGGCGGGCGTCGTCCT 1401GCTGGGCAGCCTGTTCTCGCGGAAGTAA.

Venus-Bcl-x_(L) Protein (SEQ ID NO 8) is the protein fusion encoded bythe nucleic acid of SEQ ID NO 7. The underlying Bcl-x_(L) proteinsequence (233 AA) is encoded by the DNA sequence GenBank ID: 20336334from NCBI Database:

SEQ ID NO 8 1 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICT 51TGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIF 101FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN 151VYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH 201YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGVLMSQSNREL 251VVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWH 301LADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDL 351TSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKE 401MQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQE 451RFNRWFLTGMTVAGVVLLGSLFSRK*.

Example 1 An Exemplary Vector of the Invention

FIG. 1 illustrates a construct in accordance to the invention. Thestarting vector is pSP124. See V. Lumbreras, D. R. S. and S. Purton,Plant J., 14(4):441-447 (1998). Features of the vector are listed inFIG. 1, i.e. the two regions indicated in FIG. 1 to be part of thebackbone vector, pSP124.

None pSP124 sequences are preferably engineered as individual syntheticDNA fragments and strung together via restriction enzyme sites, bywell-known techniques. Alternative approaches and mixtures of approachesare available. For example, some features are optionally introduced asPCR products or “cut and pasted” from other available constructs.Typically, sequencing and/or other assays (e.g. size analysis,hybridization) are used to verify the resultant vector.

As an example, one section of the insert is created by synthesis of aregion having a BamHI site and ending with an EcoRI site(“Synthetic_(—)1”). This region provides a transcriptional enhancerregion, two LexA binding motifs, a rubisco transcriptional promoter(including the first intron of rbcS2), a YFP-Bcl-x_(L) fusion protein,and a rubisco 3′UTR. The YFP and Bcl-x_(L) coding regions were designedin this instance to reflect the preferred codon usage in algae.

Another region incorporated is prepared by high fidelity PCR andeffectively provides the p300 (HAT) gene (“Genomic PCR”). Flanking thegenomic PCR fragment are two additional regions prepared by syntheticDNA (“Synthetic_(—)2”). The region transcriptionally upstream of thep300 gene provides the LexA binding domain coding sequence downstream oftranscriptional promoters and two LexA binding sites. The Synthetic_(—)2region provides a 3′UTR. Combined, the Synthetic_(—)2 and Genomicregions create a complete transcription unit encoding a LexA-p300 fusionprotein (GEE).

Effectively, FIG. 1 and these explanations provide an example of thefeatures of a construct of the invention and illustrate methods ofcreating the features within an algae compatible plasmid. Twotranscriptional units face opposing directions and each have two LexAbinding sites, creating an opportunity for the LexA-p300 to bind at anyof four sites and affect transcription levels of either transcriptionalunit. A third transcriptional unit provides a selection marker,bleomycin-resistance.

It will be recognized by a skilled artisan that other design approachesare available, including the incorporation within the vector ofadditional or different genes incorporated for expression, differentgene expression control features, other restriction sites, change thenumber of LexA-BS, and so on, without changing the concept behind thecreation of this vector, namely to effectively increase the levels ofexpression of the genes located in vicinity of a DNA-BS, in the presenceof a GEE that recognizes/binds the BS.

Example 2 Additional Exemplary Vectors

Two vectors are constructed which are in most respects identical, butfor the presence of a GEE unit. The vectors are otherwise the same toeach other and similar to the vector of FIG. 2A. The use of thesevectors in parallel allows testing of the p300 activity and the role ofLexA in otherwise identical genetic backgrounds. The use of two vectorsalso allows for modulation of the GEE activities by such additionalengineering, for example, as addition of other genes, addition ofmultiple copies of GEE and so on.

Notably, “LexA BS” does not refer to any limit of the number of bindingsites; anything from one BS to many BS are possibly located at theindicated position. Practically speaking, it is unlikely to utilize morethan about 8 BS, as benefit from additional sites would be unlikely.Preferably, about 2 to 6 BS are located in the region at or near the5′end of genes desirably expressed, more preferably there are 2-4 BS.

Example 3 Characterization of GEE Efficacy with a Bidirectional Promoter

Experiment 1. Use the bidirectional construct with YFP reporter in theposition of the GOI and either one of two variants of the GEE construct:[1] in which the LexA-p300 chimeric gene is driven in the oppositedirection (FIG. 1) or [2] in which only LexA is driven in the oppositedirection which serves as a control.

Algae are transformed with the two constructs and selected onappropriate antibiotic containing selection media (e.g. media containingbleocin). After selection, 100 colonies from transformation for eachconstruct are chosen to analyze the expression of the YFP transgene byassaying mRNA expression using rtPCR, protein expression with Westernblot, and single cell fluorescence by flow cytometry and fluorescentmicroscopy. The clonal populations are passaged for 2, 4, 6, and 10generations. The frequency of high-level expression of YFP are comparedbetween the LexA-p300 and LexA only clones. The LexA-p300 GEE increasesexpression and maintains a higher level of nuclear transgene expressionover time.

Example 4 Characterization of GEE Efficacy Using Distinct Plasmids

Generate two sets of stable clones: Set one is a stable cell line withthe incorporated transgene encoding the LexA-p300 fusion (FIG. 2A) thatis then transformed with a plasmid that expresses the YFP vector (FIG.2B). Set two is a stable cell line with the incorporated transgeneencoding the LexA only (related to FIG. 2A with the exception of thep300 fusion partner) that is then transformed with a plasmid thatexpresses the YFP vector (FIG. 2B).

Select for stable cell lines and characterize the YFP expression overtime by assaying mRNA expression by rtPCR, western blot to determineprotein expression, and assay of single cell fluorescence by flowcytometry and fluorescent microscopy. The clonal populations will bepassaged for 2, 4, 6, and 10 generations. Similarly, the frequency ofhigh-level expression of YFP are compared between the LexA-p300 and LexAonly clones. The LexA-p300 GEE increases expression and maintains ahigher level of nuclear transgene expression over time.

The invention described above should be read in conjunction with theaccompanying claims and drawings. The description of embodiments andexamples enable one to practice various implementations of the inventionand they are not intended to limit the invention to the preferredembodiment, but to serve as a particular example of the invention. Thoseskilled in the art will appreciate that they may readily use theconception and specific embodiments disclosed as a basis for modifyingor designing other methods and systems for carrying out the samepurposes of the present invention.

All references, including publications, patent applications, patents,and website content cited herein are hereby incorporated by reference tothe same extent as if each reference were individually and specificallyindicated to be incorporated by reference and was set forth in itsentirety herein.

The websites mentioned herein were last visited on Oct. 30, 2010.

The use of the terms “a” and “an” and “the” and similar references inthe context of describing the invention are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The word “about,” when accompanying anumerical value, is to be construed as indicating a deviation of up toand inclusive of 10% from the stated numerical value. The use of any andall examples, or exemplary language (“e.g.” or “such as”) providedherein, is intended merely to better illuminate the invention and doesnot pose a limitation on the scope of the invention unless otherwiseclaimed. NO language in the specification should be construed asindicating any non-claimed element as essential to the practice of theinvention.

What is claimed is:
 1. A eukaryotic unicellular algae, comprising: aconstruct for enhanced gene expression in an alga cell, wherein saidconstruct further comprises a eukaryotic unicellular algae compatibletranscriptional promoter functionally upstream of a coding sequence fora gene expression enhancer (GEE) fusion protein, wherein the fusionprotein comprises an algae derived p300 functionally fused to the DNAbinding protein, wherein at least the portion of the coding sequence ofthe DNA binding protein domain is codon optimized for improvedexpression in a eukaryotic unicellular algae; at least one transgenefunctionally downstream of a eukaryotic unicellular algae compatibletranscriptional promoter; and at least one DNA region that is a bindingsite for the DNA binding protein, in vicinity of at least one of saidtranscriptional promoters.
 2. The eukaryotic unicellular algae of claim1, wherein the DNA binding protein is LexA DNA Binding domain.
 3. Theeukaryotic unicellular algae of claim 1, wherein the p300 part of theGEE fusion protein is from Chlamydomonas reinhardtii.
 4. The eukaryoticunicellular algae of claim 1, wherein only a HAT domain of the p300protein is part of the GEE fusion protein.
 5. The eukaryotic unicellularalgae of claim 1, wherein the transgene is codon modified for improvedexpression in algae.
 6. The eukaryotic unicellular algae of claim 5,wherein the transgene is a fluorescence-Bcl-x_(L) fusion gene.
 7. Theeukaryotic unicellular algae of claim 6, wherein thefluorescence-Bcl-x_(L) fusion gene is a YFP-Bcl-x_(L) fusion.
 8. Theeukaryotic unicellular algae of claim 6, wherein thefluorescence-Bcl-x_(L) fusion gene is a Venus-Bcl-x_(L) fusion.
 9. Theeukaryotic unicellular algae of claim 1, further comprising at least oneselective marker.
 10. The eukaryotic unicellular algae of claim 9,wherein the GEE fusion protein and the at least one transgene areintroduced into the system on one vector and structurally arranged to beexpressed from one bidirectional promoter region and comprising DNAbinding sites in the vicinity of both promoters.
 11. The eukaryoticunicellular algae of claim 9, wherein the GEE fusion protein and thetransgene are introduced in the system on separate vectors, eachcomprising a selective marker and the selective markers are not thesame.
 12. The eukaryotic unicellular algae of claim 1, wherein the algaecompatible transcriptional promoters are hsp70, rbcS, nitA, tubA2 or acombination thereof.
 13. The eukaryotic unicellular algae of claim 1,wherein the GEE fusion protein comprises a DNA binding domainfunctionally fused to an algae derived p300 homologue having at least80% identity over the HAT region to the p300 from Chlamydomonasreinhardtii.
 14. The eukaryotic unicellular algae of claim 13, whereinthe GEE fusion protein comprises a DNA binding domain functionally fusedto the HAT domain from an algae derived p300 homologue, the homologuehaving at least 80% identity over the HAT region to the p300 fromChlamydomonas reinhardtii.